The present invention relates to single molecule detectors, methods of manufacturing same and methods of detecting single molecules using same. More particularly, the present invention relates to detectors suited for detecting the presence, motion and/or length of one or more molecules, methods of manufacturing same and methods of detecting the presence, motion and/or length of one or more molecules at a resolution, as high as, a nanometer resolution.
Single-molecule experimental techniques provide fascinating possibilities for studying systems in which molecular individuality matters. This is particularly true when the molecule forms a part of a complex environment which substantially affects its behavior, or when the molecule itself has an intricate internal structure resulting in a complex energy landscape. Because single-molecule experiments offer unique information on macromolecular interactions and chemical dynamics, unattainable by ensemble-averaged measurements, such experiments have had a remarkable impact on many scientific and technological disciplines. Single molecule studies on biological systems have already yielded important information relevant to problems that involve macromolecular motion and confirmational dynamics, photo biology, protein folding and enzyme mechanics. Undoubtedly, single-molecule studies will remain central to the biological sciences in years to come.
Single-molecule studies, as oppose to ensemble-based experiments, refer to a class of measurements where the behavior of individual molecules is followed. One advantage of such studies is the ability to determine the distribution of molecular properties by conducting many sequential measurements, hence affording the investigation of inhomogeneous systems. Another advantage of single-molecule studies is that single-molecule trajectories are direct records of the system""s fluctuations, and as such they provide dynamical and statistical information that is hidden in ensemble-averaged results. In addition, single-molecule measurements permit real-time observation of rarely populated transients, which are otherwise difficult or impossible to capture using conventional methods.
Several basic experimental approaches and their derivatives, which allow for single-molecule experiments, are known in the art. These include scanning force microscopy (SFM), optical trapping and optical microscopy and spectroscopy. SFM and optical trapping require that an external, time-varying load be applied to the substrate to enable measurement. Although SFM may be used as an imaging tool, its functionality as such is slow. Furthermore, when used as a force sensor, SFM can reliably probe motions only parallel to the scanning tip, whereas other motions cannot be detected effectively. In addition, when used as a characterization tool, SFM has a low throughput and requires highly trained personal.
Optical techniques are comparatively fast allowing the recording of molecular trajectories down to the 10-100 millisecond time-scales, and can achieve center-position accuracy of a few nanometers.
One optical technique of single molecule detection is based on flow cytometry, where an analyte solution is delivered into a rapidly flowing sheath fluid and hydrodynamically focused into a narrow sample stream. Another approach is based on analyte movement through a capillary. In flow cytometry, a sample stream passes through the center of a probe volume defined by the diameter of the focused excitation laser beam and a spatial filter placed in the image plane of a light collecting objective. Single fluorescent molecules are detected by the bursts of photons emitted as they flow through the detection volume one-at-a-time. However, focused laser beams, can generate a notable trapping potential for sizable molecules, which may affect the accuracy of the measurements. More important since these techniques rely heavily on fluorescence, they are prone to a number of photoinduced artifacts, such as triplet trapping and photobleaching. The latter dictates that observation times have to be short. Furthermore, extracting data from fluorescent and optical trapping techniques often requires complex image analysis.
Electrostatic, or capacitive, sensing is one of the most important and oldest precision sensing mechanisms. Macroscopic capacitive sensors and transducers of many shapes have been implemented. They are used for liquid level sensing, touch sensing, key switches, light switches and proximity detection.
Electret microphones implemented in telephones and tape recorders use capacitive sensing, as do Silicone accelerometers that deploy the air bag in a car. A key feature of capacitive sensors is their ability to detect the presence of material at a distance through variation in the dielectric constant. Some of the advantages of capacitive sensors are their sensitivity, accuracy and temperature stability. Furthermore, capacitive sensors are less noisy than resistive sensors and consume very little power.
On the microscopic level, the use of capacitive sensors is increasing rapidly. Use of microscopic level capacitive sensing are known in the art, for example Sohn, L. L., Saleh, O. A., Facer, G. R., Beavis, A. J., Allan, R. S. and Notterman, D. A., PNAS 97, 10687-10690 (2000), disclosed a devise intended to measure dielectric constant using a parallel plate capacitor. However, the device disclosed by Sohn et. al. fails to have any sensitivity to position. In addition, the resolution of this device is limited by the physical size of the device, hence it lacks the capability of a single molecule detection.
Capacitive sensors capable of single molecule detection have not yet been described in the art.
There is thus a widely recognized need for, and it would be highly advantageous to have, a single molecule detector and a method of detecting a single molecule, based on capacitive sensing.
According to one aspect of the present invention there is provided a detector, for determining presence, number, length concentration, position and/or motion of at least one particle present in a fluid and having a dielectric coefficient other than a dielectric coefficient of the fluid, the detector comprising: (a) a capacitor, comprising a first conductive plate and a second conductive plate defining an inter-plate volume having a longitudinal axis; and (b) at least two electrical contacts, connecting each of the first and second conductive plates to a capacitance measuring device; the capacitor being characterized by at least one variable parameter so as to allow determination and/or monitoring of presence, number, length concentration, position and/or at least one motion characteristic of the at least one particle placed within the inter-plate volume of the capacitor.
According to another aspect of the present invention there is provided A motion detection method comprising placing at least one particle present in a fluid and having a dielectric coefficient other than a dielectric coefficient of the fluid, within an inter-plate volume of a capacitor, being characterized by at least one variable parameter, and determining and/or monitoring presence, number, length concentration, longitudinal position and/or at least one motion characteristic of the at least one conductive particle, by determining a change in capacitance of the capacitor.
According to yet another aspect of the present invention there is provided a particle presence, number, length or concentration detector comprising; (a) a capacitor, comprising a first conductive plate and a second conductive plate substantially parallel to the first conductive plate, the first and second conductive plates defining an interplate volume having a longitudinal axis; and (b) at least two electrical contacts, connecting each of the first and second conductive plates to a capacitance measuring device; the capacitor being designed and constructed for allowing a determination of a presence, number or concentration of particles placed within the inter-plate volume of the capacitor, the particles being present in a fluid and having a dielectric coefficient other than a dielectric coefficient of said fluid.
According to still another aspect of the present invention there is provided a method of determining the presence, number, length or concentration of particles present in a fluid and having a dielectric coefficient other than a dielectric coefficient of the fluid, the method comprising placing the conductive particles in an inter-plate volume of a parallel plates capacitor and determining the presence, number, length or concentration of the conductive particles by determining a change in capacitance of the parallel plates capacitor.
According to still an additional aspect of the present invention there is provided a detector for determining presence, number, length concentration, position and/or motion of at least one particle present in a fluid and having a dielectric coefficient other than a dielectric coefficient of the fluid, the detector comprising: (a) a variable-width capacitor, comprising a first conductive plate and a second conductive plate having a variable distance therebetween and defining an inter-plate volume of a variable-width having a longitudinal axis; and (b) at least two electrical contacts, connecting each of the first and second conductive plates to a capacitance measuring device; the variable-width capacitor being designed and constructed for determination of a longitudinal position and/or monitoring a change of position along the longitudinal axis of a single conductive particle placed within the inter-plate volume of the variable-width capacitor.
According to a further aspect of the present invention there is provided a motion detection method comprising placing at least one particle present in a fluid and having a dielectric coefficient other than a dielectric coefficient of the fluid, within an inter-plate volume of a variable-width capacitor and determining and/or monitoring a longitudinal position and/or at least one motion characteristic of the at least one conductive particle by determining a change in capacitance of the variable-width capacitor.
According to further features in preferred embodiments of the invention described below, the at least one variable parameter is selected from the group consisting of a variable dielectric coefficient and a variable cross-sectional area, the cross-sectional area being perpendicular to the longitudinal axis.
According to still further features in the described preferred embodiments the at least one, particle is self-conductive.
According to still further features in the described preferred embodiments the at least one particle is linkable to at least one conductive particle.
According to still further features in the described preferred embodiments the at least one particle is selected from the group consisting of a cell, a bacterium, a biological molecule, an organic molecule and a polymer.
According to still further features in the described preferred embodiments the determination and/or monitoring is at a nanometer resolution.
According to still further features in the described preferred embodiments the determination and/or monitoring is in a sub-microsecond time scales.
According to still further features in the described preferred embodiments the first and second conductive plates engage opposite inner-faces of a capillary.
According to still further features in the described preferred embodiments the first and second conductive plates engage opposite outer-faces of a capillary.
According to still further features in the described preferred embodiments the capillary has a profile selected from the group consisting of a polygonal profile a circular profile an ellipsoidal profile and an irregular pattern profile.
According to still further features in the described preferred embodiments the capillary is characterized by a variable cross section at any position along the longitudinal axis.
According to still further features in the described preferred embodiments the at least one motion characteristic is selected from the group consisting of velocity and acceleration.
According to still further features in the described preferred embodiments the detector further comprising at least one additional conductive layer interposted between the first and the second conductive layers, the at least one additional conductive layer having a surface area substantially smaller than a surface area of both the first and the second conductive layers.
According to still further features in the described preferred embodiments the detector further comprising at least one electrical isolating layer, covering the at least one additional conductive layer.
According to still further features in the described preferred embodiments at least one additional conductive layer is grounded.
According to still further features in the described preferred embodiments the at least one additional conductive layer is a made of Gold.
According to still further features in the described preferred embodiments the electrical isolating layer is a made of quartz.
According to still further features in the described preferred embodiments the method further comprising providing a dielectric material between the first conductive plate and the second conductive plate, the dielectric material having a dielectric coefficient.
According to still further features in the described preferred embodiments the dielectric coefficient is constant.
According to still further features in the described preferred embodiments the dielectric coefficient varies along the longitudinal axis.
According to still further features in the described preferred embodiments a transverse dimension of the first and the second conductive plates, with respect to the longitudinal axis, is constant along the longitudinal axis.
According to still further features in the described preferred embodiments a transverse dimension of the first and the second conductive plates, with respect to the longitudinal axis, varies along the longitudinal axis.
According to still further features in the described preferred embodiments the step of positioning the first conductive plate and the second conductive plate comprises: (a) providing a pullable tube having a profile; (b) pulling the tube at a controlled rate so as to provide a capillary having a predetermined maximal diameter; and (c) applying the first and the second conductive plates on opposite faces of the capillary.
According to still further features in the tie described preferred embodiments the opposite faces are selected from the group consisting of opposite inner-faces and opposite outer-faces.
According to still further features in the described preferred embodiments the step of applying is effected from a procedure selected from the group consisting of evaporation, lift-off, shadow-evaporation, nano-manipulation and focused ion milling.
According to still further features in the described preferred embodiments the profile is selected from the group consisting of a polygonal profile a circular profile an ellipsoidal profile and an irregular pattern profile.
According to still further features in the described preferred embodiments the step of pulling the tube substantially retain the profile.
According to still further features in the described preferred embodiments the step of pulling the tube is done by a micropipette puller.
According to still further features in the described preferred embodiments the micropipette puller is a laser based micropipette puller.
According to still further features in the described preferred embodiments the laser is a CO2 laser.
According to still further features in the described preferred embodiments the method further comprising the step of varying a diameter of the profile along the longitudinal axis, during or subsequent to the step of pulling the tube.
According to still further features in the described preferred embodiments the method further comprising the steps of applying at least one additional conductive layer onto the capillary, and covering the at least one additional conductive layer by an electrical isolating layer, prior to the step (c).
According to still further features in the described preferred embodiments the method further comprising grounding the at least one additional conductive layer.
According to still further features in the described preferred embodiments the at least one additional conductive layer is a made of a material selected from the group consisting of Gold and Aluminium.
According to still further features in the described preferred embodiments the covering is effected from a procedure selected from the group consisting of evaporation, lift-off, shadow-evaporation, nano-manipulation and focused ion milling.
According to still further features in the described preferred embodiments the nano-manipulation is done by atomic force microscope.
According to an additional aspect of the present invention there is provided a method of manufacturing a motion detector for detecting at least one conductive particle, the method comprising the steps of: positioning a first conductive plate and a second conductive plate such that the second conductive plate is spaced apart from the first conductive plate, so as to define an inter-plate volume having a longitudinal axis; and providing a at least two electrical contacts, connecting each of the first and the second conductive plates to a capacitance measuring device.
According to yet an additional aspect of the present invention there is provided a method of manufacturing a motion detector for detecting at least one conductive particle, the method comprising the steps of: (a) etching a non conductive substrate so as to provide at least two channels; (b) coating the at least two channels by a conductive material so as to provide at least two coated channels; and (d) providing a at least two electrical contacts, connecting each of the first and the second conductive plates to a capacitance measuring device.
According to further features in preferred embodiments of the invention described below, the capacitance measuring device is selected from the group consisting of a capacitance meter and a capacitance bridge.
According to still further features in the described preferred embodiments the capacitance measuring device is configured and designed to allow measuring of capacitance at a 1xc3x9710xe2x88x9218 F resolution.
According to still further features in the described preferred embodiments the capacitance measuring device is operable to measure a time dependence of a change in capacitance.
According to still further features in the described preferred embodiments the particles arc dissolved in the fluid.
According to still further features in the described preferred embodiments the particles are dispersed in the fluid.
According to still further features in the described preferred embodiments the first and the second conductive plates are made of a material selected from the group consisting of Gold and Aluminium.
According to still further features in the described preferred embodiments the non conductive substrate is made of poly-Si.
According to still further features in the described preferred embodiments the method further comprising positioning the non conductive substrate onto a conductive substrate, prior to the step of etching.
According to still further features in the described preferred embodiments the conductive substrate is a doped Si wafer.
According to still further features in the described preferred embodiments the method further comprising grounding the conductive substrate.
According to still further features in the described preferred embodiments the step of providing the at least two electrical contacts is done by patterning and evaporation.
According to still further features in the described preferred embodiments the patterning is effected by at least one procedure selected from the group consisting of photolithography and lift-off technique.
According to still further features in the described preferred embodiments the electrical contacts comprise bonding pads.
According to still further features in the described preferred embodiments the method further comprising etching at least two reservoirs in the non conductive substrate prior to the stop of coating.
According to still further features in the described preferred embodiments the method further comprising covering the first and the second conductive plates by a non conductive slip having at least two holed so as to allow liquid passage therethrough into the at least two reservoirs.
According to still further features in the described preferred embodiments the conductive material is selected from the group consisting of Gold and Aluminium.
The present invention successfully addresses the shortcomings of the presently known configurations by providing a detector, for determining presence, number, length concentration, position and/or motion of at least one particle. The detector enjoys properties far exceeding those characterizing prior art detectors.
Implementation of the method and system of the present invention involves performing or completing selected tasks or steps manually, automatically, or a combination thereof. Moreover, according to actual instrumentation and equipment of preferred embodiments of the method and system of the present invention, several selected steps could be implemented by hardware or by software on any operating system of any firmware or a combination thereof. For example, as hardware, selected steps of the invention could be implemented as a chip or a circuit. As software, selected steps of the invention could be implemented as a plurality of software instructions being executed by a computer using any suitable operating system. In any case, selected steps of the method and system of the invention could be described as being performed by a data processor, such as a computing platform for executing a plurality of instructions.